Environmental Science and Pollution Research

, Volume 22, Issue 6, pp 4555–4565 | Cite as

A spectroscopic study on U(VI) biomineralization in cultivated Pseudomonas fluorescens biofilms isolated from granitic aquifers

  • Evelyn Krawczyk-BärschEmail author
  • Laura Lütke
  • Henry Moll
  • Frank Bok
  • Robin Steudtner
  • André Rossberg
Research Article


The interaction between the Pseudomonas fluorescens biofilm and U(VI) were studied using extended X-ray absorption fine structure spectroscopy (EXAFS), and time-resolved laser fluorescence spectroscopy (TRLFS). In EXAFS studies, the formation of a stable uranyl phosphate mineral, similar to autunite (Ca[UO2]2[PO4]2•2–6H2O) or meta-autunite (Ca[UO2]2[PO4]2•10–12H2O) was observed. This is the first time such a biomineralization process has been observed in P. fluorescens. Biomineralization occurs due to phosphate release from the cellular polyphosphate, likely as a cell’s response to the added uranium. It differs significantly from the biosorption process occurring in the planktonic cells of the same strain. TRLFS studies of the uranium-contaminated nutrient medium identified aqueous Ca2UO2(CO3)3 and UO2(CO3)3 4− species, which in contrast to the biomineralization in the P. fluorescens biofilm, may contribute to the transport and migration of U(VI). The obtained results reveal that biofilms of P. fluorescens may play an important role in predicting the transport behavior of uranium in the environment. They will also contribute to the improvement of remediation methods in uranium-contaminated sites.


Pseudomonas fluorescens Biofilm Uranium Meta-autunite EXAFS TRLFS 



This work was funded by the Federal Ministry of Economics and Technology (BMWi) under contract number 02E10618. The X-ray absorption spectroscopy (XAS) measurements were performed at BM20 (ROBL) at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France). In particular, thanks are given to Andreas Scheinost and Christoph Hennig (ROBL Group, ESRF, Grenoble, France) for their support during the XAS measurements and their help in evaluating the data. We thank Ursula Schaefer, Aline Ritter, and Carola Eckardt for the analysis. Stephan Weiß′s skilful work on sample preparation for EXAFS is gratefully acknowledged. Nina Huittinen is thanked for the fruitful discussion.


  1. Allison DG, Ruiz B, SanJose C, Jaspe A, Gilbert P (1998) Extracellular products as mediators of the formation and detachment of Pseudomonas fluorescens biofilms. FEMS Microbiol Lett 167:179–184CrossRefGoogle Scholar
  2. Ankudinov AL, Ravel B, Rehr JJ, Conradson SD (1998) Real-space multiple-scattering calculation and interpretation of x-ray-absorption near-edge structure. Phys Rev B 58:7565CrossRefGoogle Scholar
  3. Beazley MJ, Martinez RJ, Sobecky PA, Webb SM, Taillefert M (2007) Uranium biomineralization as a result of bacterial phosphatase activity: insights from bacterial isolates from a contaminated subsurface. Environ Sci Technol 41:5701–5707CrossRefGoogle Scholar
  4. Bencheikh-Latmani R, Leckie JO (2003) Association of uranyl with the cell wall of Pseudomonas fluorescens inhibits metabolism. Geochim Cosmochim Acta 67(21):4057–4066CrossRefGoogle Scholar
  5. Bernhard G, Geipel G (2007) Bestimmung der Bindungsform des Urans in Mineralwässern. Vom Wasser 105(3):7–10Google Scholar
  6. Bernhard G, Geipel G, Brendler V, Nitsche H (1996) Speciation of uranium in seepage waters from a mine tailing pile studied by time-resolved laser-induced fluorescence spectroscopy (TRLFS). Radiochim Acta 74:87–91Google Scholar
  7. Costerton JW, Lewandowski Z, Caldwell DE, Korber DR, Lappin-Scott HM (1995) Microbial biofilms. Annu Rev Microbiol 49:711–745CrossRefGoogle Scholar
  8. De Beer D, Stoodley P, Roe F, Lewandowski Z (1994) Effects of biofilm structures on oxygen distribution and mass transport. Biotechnol Bioeng 43:1131–1138CrossRefGoogle Scholar
  9. Flemming H-C (1991) Biofilme und Wassertechnologie. Teil 1: Entstehung, Aufbau, Zusammensetzung und Eigenschaften von Biofilmen. gwf-Wasser/Abwasser 132(4):197–207Google Scholar
  10. Francis AJ, Gillow JB, Dodge CJ (1998) Role of bacteria as biocolloids in the transport of actinides from a deep underground radioactive waste repository. Radiochim Acta 82:347–354Google Scholar
  11. Francis AJ, Gillow JB, Dodge CJ, Harris R, Beveridge TJ, Papenguth HW (2004) Uranium association with halophilic and non-halophilic bacteria and archaea. Radiochim Acta 92:481–488CrossRefGoogle Scholar
  12. Geipel G (2006) Some aspects of actinide speciation by laser induced spectroscopy. Coord Chem Rev 250:844–854CrossRefGoogle Scholar
  13. Geipel G, Thieme M, Bernhard G, Nitsche H (1994) Distribution of uranium and radionuclides in a uranium-mining rockpile in schlema, Saxony, Germany. Radiochim Acta 66(67):305–308Google Scholar
  14. Geissler A, Selenska-Pobell (2005) Addition of U(VI) to a uranium mining waste sample and resulting changes in the indigenous bacterial community. Geobiology 3:275–285Google Scholar
  15. Grenthe I, Fuger J, Konings RJM, Lemire RJ, Muller AJ, Nguyen-Trung C, Wanner H (1992) Chemical thermodynamics of uranium. Elsevier, AmsterdamGoogle Scholar
  16. Guillaumont R, Fanghänel T, Fuger J, Grenthe I, Neck V, Palmer DA, Rand MH (2003) Update on the chemical thermodynamics of uranium, neptunium, plutonium, americium and technetium. Chemical Thermodynamics 5. (ed. OECD Nuclear Energy Agency). Elsevier, AmsterdamGoogle Scholar
  17. Harrison JJ, Ceri H, Stremick CA, Turner RJ (2004) Differences in biofilm and planktonic cell mediated reduction of metalloid oxyanions. FEMS Microbiol Lett 235:357–362CrossRefGoogle Scholar
  18. Hudson EA, Allen PG, Terminello LJ, Denecke MA, Reich T (1996) Polarized X-ray-absorption spectroscopy of uranyl ion: comparison of experiment and theory. Phys Rev B 54:156–165CrossRefGoogle Scholar
  19. Jerden JL, Sinha AK (2003) Phosphate based immobilization of uranium in an oxidizing bedrock aquifer. Appl Geochem 18:823–843CrossRefGoogle Scholar
  20. Jroundi F, Merroun ML, Arias JM, Rossberg A, Selenska-Pobell S, González-Munoz MT (2007) Spectroscopic and microscopic characterization of uranium biomineralization in Myxococcus xanthus. Geomicrobiol J 24:441–449CrossRefGoogle Scholar
  21. Kalmykov SN, Choppin GR (2000) Mixed Ca2+/UO2 2+/CO3 2− complex formation at different ionic strengths. Radiochim Acta 88:603–606CrossRefGoogle Scholar
  22. Krawczyk-Bärsch E, Lünsdorf H, Arnold T, Brendler V, Eisbein E, Jenk U, Zimmermann U (2011) The influence of biofilms on the migration of uranium in acid mine drainage (AMD) waters. Sci Total Environ 409:3059–3065CrossRefGoogle Scholar
  23. Krawczyk-Bärsch E, Lünsdorf H, Pedersen K, Arnold T, Brendler V, Bok F, Steudtner R, Lehtinen A, Brendler V (2012) Immobilization of uranium in biofilm microorganisms exposed to groundwater seeps over granitic rock tunnel walls in Olkiluoto, Finland. Geochim Cosmochim Acta 96:94–104CrossRefGoogle Scholar
  24. Krueger S, Olson GJ, Johnsonbaugh D, Beveridge TJ (1993) Appl Environ Microbiol 59:4056–4064Google Scholar
  25. Lawrence JR, Korber DR, Hoyle BD, Costerton JW, Caldwell DE (1991) Optical sectioning of microbial biofilms. J Bacteriol 173:6558–6567Google Scholar
  26. Lloyd JR, Macaskie LE (2002) Biochemical basis of microbe-radionuclide interactions. In: Keith-Roach MJ, Livens FR (eds) Interactions of microorganisms with radionuclides. Elsevier Sciences, Oxford, pp 313–342CrossRefGoogle Scholar
  27. Lütke L, Moll H, Bernhard G (2012) Insights into the uranium(VI) speciation with Pseudomonas fluorescens on a molecular level. Dalton Trans 41:13370–13378CrossRefGoogle Scholar
  28. Makarov ES, Ivanov VI (1960) The crystalline structure of Ca(UO2)2(PO4)2•6H2O meta-autunite. Dokl Akad Nauk SSSR 132(3):673–676Google Scholar
  29. Martinez RJ, Beazley MJ, Taillefert M, Arakaki AK, Skolnick J, Sobecky PA (2007) Aerobic U(VI) bioprecipitation by metal-resistant bacteria isolated from radionuclide- and metal-contaminated subsurface soils. Environ Microbiol 9(12):3122–3133CrossRefGoogle Scholar
  30. Matz W, Schell N, Bernhard G, Prokert F, Reich T, Claussner J, Oehme W, Schlenk R, Dienel S, Funke H, Eichhorn F, Betzl M, Prohl D, Strauch U, Hüttig G, Krug H, Neumann W, Brendler V, Reichel P, Denecke MA, Nitsche H (1999) ROBL - a CRG beamline for radiochemistry and materials research at the ESRF. J Synchrotron Radiat 6:1076CrossRefGoogle Scholar
  31. Merroun ML, Selenska-Pobell S (2008) Bacterial interactions with uranium: an environmental perspective. J Contam Hydrol 102:285–295CrossRefGoogle Scholar
  32. Merroun ML, Hennig C, Rossberg A, Reich T, Selenska-Pobell S (2003) Characterization of U(VI)-Acidithiobacillus ferrooxidans complexes using EXAFS, transmission electron microscopy, and energy-dispersive X-ray analysis. Radiochim Acta 91:583–591CrossRefGoogle Scholar
  33. Merroun ML, Raff J, Rossberg A, Hennig C, Reich T, Selenska-Pobell S (2005) Complexation of uranium by cells and S-layer sheets of bacillus sphaericus JG-A12. Appl Environ Microbiol 71(9):5532–5543CrossRefGoogle Scholar
  34. Merroun ML, Nedelkova M, Rossberg A, Hennig C, Selenska-Pobell S (2006) Interaction mechanisms of bacterial strains isolated from extreme habitats with uranium. Radiochim Acta 94:723–729CrossRefGoogle Scholar
  35. Moulin C, Beaucaire C, Decambox P, Mauchien P (1990) Determination of uranium in solution at the ng L-1 level by time resolved laser-induced spectrofluorimetry—application to geological samples. Anal Chim Acta 238(2):291–296CrossRefGoogle Scholar
  36. Moulin C, Decambox P, Moulin V, Decaillon JG (1995) Uranium speciation in solution by time-resolved laser-induced fluorescence. Anal Chem 67(2):348–353CrossRefGoogle Scholar
  37. Nazina TN, Luk′yanova EA, Zakharova EV, Konstantinova LI, Kalmykov SN, Poltaraus AB, Zubkov AA (2010) Microorganisms in a disposal site for liquid radioactive wastes and their influence on radionuclides. Geomicrobiol J 27:473–486CrossRefGoogle Scholar
  38. Nedelkova M, Merroun ML, Rossberg A, Hennig C, Selenska-Pobell S (2007) Microbacterium isolates from the vicinity of a radioactive waste depository and their interactions with uranium. FEMS Microbiol Ecol 59:694–705CrossRefGoogle Scholar
  39. Neu MP, Boukhalfa H, Merroun ML (2010) Biomineralization and biotransformations of actinide materials. MRS Bull 35:849–857CrossRefGoogle Scholar
  40. O’Toole GA, Kolter R (1998) Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signaling pathways: a genetic analysis. Mol Microbiol 28(3):449–461CrossRefGoogle Scholar
  41. Patel TD, Bott TR (1991) Oxygen diffusion through a developing biofilm of Pseudomonas fluorescens. J Chem Technol Biotechnol 52:87–199Google Scholar
  42. Pedersen K (2005) Microorganisms and their influence on radionuclide migration in igneous rock environments. J Nucl Radiochem Sci 6(1):11–15CrossRefGoogle Scholar
  43. Pons MP, Fusté MC (1993) Uranium uptake by immobilized cells of Pseudomonas strain EPS 5028. Appl Microbiol Biotechnol 39:661–665CrossRefGoogle Scholar
  44. Radeva G, Selenska-Pobell S (2005) Bacterial diversity in water samples from uranium wastes as demonstrated by 16S rDNA and ribosomal intergenic spacer amplification retrievals. Can J Microbiol 51:910–923CrossRefGoogle Scholar
  45. Ressler T (1998) WinXAS: a program for X-ray absorption spectroscopy data analysis under MS-Windows. J Synchrotron Radiat 5:118CrossRefGoogle Scholar
  46. Robertson M, Hapca SM, Moshynets O, Spiers A (2013) Air-liquid interface biofilm formation by psychrotrophic pseudomonads recovered from spoilt meat. Antonie Van Leeuwenhoek 103:251–259CrossRefGoogle Scholar
  47. Späth R, Flemming H-C, Wuertz S (1998) Sorption properties of biofilms. Water Sci Technol 37:207–210CrossRefGoogle Scholar
  48. Steudtner R, Sachs S, Schmeide K, Brendler V, Bernhard G (2011) Ternary uranium(VI) carbonato humate complex studied by cryo-TRLFS. Radiochim Acta 99:687–692CrossRefGoogle Scholar
  49. Stoodley P, deBeer D, Lewandowski Z (1994) Liquid flow in biofilm systems. Appl Environ Microbiol 60:2711–2716Google Scholar
  50. Teitzel GM, Parsek MR (2003) Heavy metal resistance of biofilm and planktonic Pseudomonas aeruginosa. Appl Environ Microbiol 69:2313–2320CrossRefGoogle Scholar
  51. Ude S, Arnold DL, Moon CD, Timms-Wilson T, Spiers AJ (2006) Biofilm formation and cellulose expression among diverse environmental Pseudomonas isolates. Environ Microbiol 8(11):1997–2011CrossRefGoogle Scholar
  52. van Hullebusch ED, Zandvoort MH, Lens PNL (2003) Metal immobilisation by biofilms: mechanisms and analytical tools. Rev Environ Sci Biotechnol 2:9–33CrossRefGoogle Scholar
  53. Webb SM (2005) Sixpack: a graphical user interface for XAS analysis using IFEFFIT. Phys Scr T115:1011–1014CrossRefGoogle Scholar
  54. Workentine ML, Harrison JJ, Pernilla PU, Stenroos U, Ceri H, Turner RJ (2008) Pseudomonas fluorescens′ view of the periodic table. Environ Microbiol 10(1):238–250Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Evelyn Krawczyk-Bärsch
    • 1
    Email author
  • Laura Lütke
    • 1
  • Henry Moll
    • 1
  • Frank Bok
    • 1
  • Robin Steudtner
    • 1
  • André Rossberg
    • 1
  1. 1.Helmholtz-Zentrum Dresden-RossendorfInstitute of Resource EcologyDresdenGermany

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